Epigenetic modifiers are frequently mutated in hematopoietic diseases. Understanding the functional and molecular consequences of these mutations is important for determining how the mutation manifests the disease, as well as allowing us to understand the cell fate decisions that underlie normal hematopoiesis. Recent data suggests that Kdm6b, the demethylase responsible for removing the histone H3 lysine 27 trimethylation mark (H3K27me3), is dysregulated in a myriad of blood disorders and cancers. Our evidence also suggests Kdm6b influences hematopoietic stem cell (HSC) fate decisions through the regulation of bivalent gene expression, which upon ablation of the DNA methyltransferase enzyme Dnmt3a becomes unbalanced. This suggests that Kdm6b is an important epigenetic regulator of hematopoiesis; however, to date no studies have investigated the role of Kdm6b in HSC function. We aim to elucidate the role of Kdm6b in normal hematopoiesis in order to better understand how dysregulation of this epigenetic modifier contributes to hematopoietic disease. Using genetic mouse models, we will determine the role of Kdm6b in normal hematopoiesis through classical bone marrow transplantation methods such as competitive HSC transplants and limiting dilution assays. In order to determine the downstream targets of Kdm6b in vivo, we will use microarray and ChIP-qPCR assays on HSCs and downstream progenitors. Further, in vitro analysis will be performed on a genome-wide scale by ChIP-SEQ using an endogenously tagged cell line to explore Kdm6b specificity to bivalent domains. Lastly, we will determine if controlling Kdm6b expression in the absence of Dnmt3a resolves HSC function by using genetic mouse models and competitive HSC serial transplantations. Hypothesis: Kdm6b is essential for long-term maintenance and differentiation of HSCs, and required for normal hematopoiesis. Aim 1: Characterize the role of Kdm6b in normal hematopoiesis and HSC self-renewal. Aim 2: Identify the molecular function and downstream targets of Kdm6b in hematopoietic cells. Aim 3: Determine if Dnmt3a-null HSCs can be rescued through reducing Kdm6b expression levels.